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Retron Eco2 breaks tRNAs for anti-phage defense

ABSTRACT: Retrons mediate bacterial anti-phage defense by reverse transcribing non-coding RNA into multicopy single-stranded DNA (msDNA). Our study reveals the unique trimeric nucleoprotein structure of Eco2, which solely relies on a single protein for defense, unlike most other retrons. This structure supports an msDNA-dependent regulation of the Eco2's reverse-transcriptase and TOPRIM/RNase H (TR) fusion protein. We also show that Eco2's broad defense against various phages is triggered by a phage-encoded endonuclease that degrades the msDNA. msDNA decay in turn activates the TR domain for tRNA cleavage, resulting in shutdown of gene expression for abortive infection. Our findings not only advance the understanding of Eco2’s biogenesis and defense mechanism but also provide a structural basis for engineering of this system.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Wieland Steinchen

LAB HEAD: Gert Bange

PROVIDER: PXD060313 | Pride | 2026-02-03

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	240829_HX_067_Mi_Apo.zip	Other
	240829_HX_067_Mi_Eco2.DnX	Other
	240829_HX_067_Mi_PLGS.zip	Other
	240829_HX_067_Mi_msDNA.zip	Other
	DescriptionOfRawDataFiles.xlsx	Xlsx

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Project description:Enzymes play a crucial role in biological systems and biotechnological processes as vital and sustainable catalysts that facilitate and regulate biochemical reactions. Inhibition of product formation of an enzyme by its own substrate was originally dismissed as an artifact of biochemical analysis, but is now a proven fact and occurs in about 20% of known enzymes1. Substrate inhibition is attributed to the formation of an unproductive enzyme-substrate complex with no structural evidence of unproductivity provided to date1–6. Here we show in detail the molecular mechanism of substrate inhibition of the tobacco glucosyltransferase NbUGT72AY1, which transfers glucose from its nucleotide donor substrate to phenol acceptors as a plant protection measure. The peculiarity that the effector β-carotene strongly attenuates the substrate inhibition of NbUGT72AY1, although it unexpectedly acts as a competitive acceptor-substrate inhibitor, allowed us to uncover the conformational changes that occur during substrate binding in the active and substrate-inhibited complexes of this protein. X-ray crystallography revealed structurally different ternary enzyme-substrate complexes that do not conform to the classical compulsory ordered or random mechanism of an enzyme-catalyzed sequential bi-substrate reaction. We suggest an alternative pathway in which the two substrates bind randomly to the enzyme, but the reaction is only catalyzed if a specific order of substrate binding is observed (asymmetric cooperativity). The new paradigm explains substrate inhibition in monomeric multi-substrate enzymes and reactivation of activity by competitive inhibitors. This alternative concept opens up new avenues of research in metabolic regulation as well as a wealth of novel industrial applications. The results enable novel approaches, e.g. to minimize the effects of food components on drug metabolism and to study plant defense mechanisms from a new perspective and offer alternative approaches for the design of more efficient production processes.

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Retron Eco2 breaks tRNAs for anti-phage defense

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